Role of subchondral bone properties and changes in development of load-induced osteoarthritis in mice.

OBJECTIVE: Animal models recapitulating post-traumatic osteoarthritis (OA) suggest that subchondral bone (SCB) properties and remodeling may play major roles in disease initiation and progression. Thus, we investigated the role of SCB properties and its effects on load-induced OA progression by applying a tibial loading model on two distinct mouse strains treated with alendronate (ALN). DESIGN: Cyclic compression was applied to the left tibia of 26-week-old male C57Bl/6 (B6, low bone mass) and FVB (high bone mass) mice. Mice were treated with ALN (26 µg/kg/day) or vehicle (VEH) for loading durations of 1, 2, or 6 weeks. Changes in articular cartilage and subchondral and epiphyseal cancellous bone were analyzed using histology and microcomputed tomography. RESULTS: FVB mice exhibited thicker cartilage, a thicker SCB plate, and higher epiphyseal cancellous bone mass and tissue mineral density than B6 mice. Loading induced cartilage pathology, osteophyte formation, and SCB changes; however, lower initial SCB mass and stiffness in B6 mice did not attenuate load-induced OA severity compared to FVB mice. By contrast, FVB mice exhibited less cartilage damage, and slower-growing and less mature osteophytes. In B6 mice, inhibiting bone remodeling via ALN treatment exacerbated cartilage pathology after 6 weeks of loading, while in FVB mice, inhibiting bone remodeling protected limbs from load-induced cartilage loss. CONCLUSIONS: Intrinsically lower SCB properties were not associated with attenuated load-induced cartilage loss. However, inhibiting bone remodeling produced differential patterns of OA pathology in animals with low compared to high SCB properties, indicating that these factors do influence load-induced OA progression.

Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes.

OBJECTIVE: To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. METHODS: Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. RESULTS: Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CONCLUSIONS: CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13.

Phlpp1 facilitates post-traumatic osteoarthritis and is induced by inflammation and promoter demethylation in human osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and a leading cause of disability. OA is characterized by articular chondrocyte deterioration, subchondral bone changes and debilitating pain. One strategy to promote cartilage regeneration and repair is to accelerate proliferation and matrix production of articular chondrocytes. We previously reported that the protein phosphatase Phlpp1 controls chondrocyte differentiation by regulating the activities of anabolic kinases. Here we examined the role of Phlpp1 in OA progression in a murine model. We also assessed PHLPP1 expression and promoter methylation. DESIGN: Knee joints of WT and Phlpp1(-/-) mice were surgically destabilized by transection of the medial meniscal ligament (DMM). Mice were assessed for signs of OA progression via radiographic and histological analyses, and pain assessment for mechanical hypersensitivity using the von Frey assay. Methylation of the PHLPP1 promoter and PHLPP1 expression were evaluated in human articular cartilage and chondrocyte cell lines. RESULTS: Following DMM surgeries, Phlpp1 deficient mice showed fewer signs of OA and cartilage degeneration. Mechanical allodynia associated with DMM surgeries was also attenuated in Phlpp1(-/-) mice. PHLPP1 was highly expressed in human articular cartilage from OA patients, but was undetectable in cartilage specimens from femoral neck fractures (FNFxs). Higher PHLPP1 levels correlated with less PHLPP1 promoter CpG methylation in cartilage from OA patients. Blocking cytosine methylation or treatment with inflammatory mediators enhanced PHLPP1 expression in human chondrocyte cell lines. CONCLUSION: Phlpp1 deficiency protects against OA progression while CpG demethylation and inflammatory cytokines promote PHLPP1 expression.

PTEN ELEVATION, AUTOPHAGY AND METABOLIC REPROGRAMMING MAY BE INDUCED IN HUMAN CHONDROCYTES DURING STEROIDS OR NUTRIENT DEPLETION AND OSTEOARTHRITIS.

The exact mechanisms controlling the development and progression of osteoarthritis have not yet been clarified. Our aim was to investigate new pathomechanisms, with an emphasis on novel molecular targets that might regulate human chondrocytes in osteoarthritis. As a model for studying cell survival and metabolism, C-28/I2 and T/C-28a4 human chondrocytes were grown in complete medium, in dex-tran-coated charcoal treated medium and in serum-free medium. Healthy and osteoarthritic human cartilage samples were obtained from discarded surgical material. Cell survival, PTEN, AKT, Beclin1, AMBRA, AMPK and glucose/triglyceride metabolism were evaluated by immunoblotting and spectro-photometric assays. Starvation and steroids depletion decreased cell survival concomitantly with PTEN elevation, repression of the PI3K/AKT signaling axis and autophagy activation. These experimental conditions promoted the accumulation of glucose, decreased levels of G6PDH and resulted in differen-tial expression of OXPHOS complexes. Furthermore, they induced the expression of AMPK, reduced triglyceride levels and increased lipase activity, which was accompanied by a change in chondrocytes toward a fibroblast-like morphology. In osteoarthritic human cartilage, increased PTEN, AMPK and autophagy reflected the chondrocyte responses observed during starvation and steroids depletion. In conclusion, we defined the metabolic phenotype of human chondrocytes, in which both starvation and steroids depletion induce the activation of PTEN, AMPK and autophagy signaling, concomitant with metabolic reprogramming. Our data may aid in the development of novel in vitro models for the discovery and design of drugs or nutraceuticals capable of ameliorating the course of osteoarthritis.

Biochemical evidence for gap junctions and Cx43 expression in immortalized human chondrocyte cell line: a potential model in the study of cell communication in human chondrocytes.

OBJECTIVE: The development of chondrocytic cell lines has enabled the investigation of the role of cellular phenotype and mechanisms in articular cartilage biology and physiopathology of several rheumatic diseases. Among them, the T/C-28a2 cell line has become a common tool in cartilage research. Recent results from our group have revealed that primary human chondrocytes in tissue and in monolayer culture contain high levels of connexin 43 (Cx43) and are able to directly communicate through gap junction (GJ) channels. These results challenge the existing thesis of cartilage physiology, that chondrocytes do not have the capacity to physically communicate with each other. Established cell lines offer the advantage of convenience and uniformity; however, the establishment process may cause a disruption of GJ. This study was performed to investigate if T/C-28a2 cells contain Cx43 protein and form functional channels. METHODS: Cx43 was characterized by RT-qPCR, Western blotting, and immunohistochemistry (IHC). Electrophysiology experiments, Lucifer Yellow (LY) uptake, electroporation in situ and scrape loading assay were performed to test the functionality of GJs. RESULTS: T/C-28a2 cells express Cx43. Electrophysiology experiments and LY uptake confirmed the capacity of these cells to communicate through GJ channels, although these cells contain significant levels of active c-Src kinase, presumably due to their immortalization with the Simian Virus 40 large T antigen. The results were validated using primary chondrocytes (PC). CONCLUSIONS: These results reveal that the T/C-28a2 line may provide a useful in vitro model for the study of Cx43 function and cell communication to understand the physiology of chondrocytes and cartilage.

Oxidative stress and status of antioxidant enzymes in children with Kashin-Beck disease.

OBJECTIVES: To clarify whether there is oxidative stress in Kashin-Beck disease (KBD) and if cartilage damage from reactive oxygen species (ROS) and oxidative stress mediate the chondral necrosis in articular cartilage of KBD. METHODS: We recruited 64 KBD patients, 46 healthy children from severely affected KBD regions, 81 healthy children from a non-severely affected KBD endemic regions, and 91 healthy control children from a non-KBD region. Ten patients with KBD from the non-severely affected KBD regions were included in the experiment. The 2,3-DAN fluorescence technique was used to test selenium in the hair and blood. The biochemical techniques used to test the indicators of oxidative stress included thiobarbituric acid reactive substances (TBARS) levels, and antioxidant enzyme activities in serum samples. Histochemical staining was used to detect proteoglycans in cartilage sections. The 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanisine (8-OHdG) were localized by immunohistochemistry. RESULTS: The levels of TBARS in serum were significantly increased in KBD children. The levels of antioxidants in serum were significantly higher in both KBD and normal children from KBD regions than in the normal children from non-KBD regions. The percentage of chondrocytes staining for 4-HNE and 8-OHdG in KBD patients was significantly higher than in controls. Staining for 4-HNE and 8-OHdG in KBD patients was prominent in all zones of articular cartilage, especially in the necrotic chondrocytes of the deep zone. CONCLUSION: KBD is an oxidative stress-related disease, and the oxidative stress in cartilage contributes to the pathology of cartilage damage in KBD.

Acute inflammation with induction of anaphylatoxin C5a and terminal complement complex C5b-9 associated with multiple intra-articular injections of hylan G-F 20: a case report.

OBJECTIVE: The purpose of this case report was to investigate local immune mechanisms present during an acute inflammatory flare initiated by viscosupplementation with hylan G-F 20 in a patient with osteoarthritis (OA) and past meniscectomy. EXPERIMENTAL DESIGN: A patient with a history of bilateral OA and partial left knee meniscectomy, who had received three injections of hylan G-F 20, was diagnosed with an acute flare reaction in the left knee. Her chart was evaluated for clinical, radiological, and laboratory findings and for clinical follow-up. Histopathological synovial examination and real-time polymerase chain reaction (RT-PCR) for genes with major roles in local inflammation and enzyme-linked immunosorbent assays (ELISAs) for markers of complement activation and cytokines were performed. To study the impact of the inflammatory and immune features we compared the case patient with groups of three representative OA and three rheumatoid arthritis (RA) patients. RESULTS: The patient exhibited evidence of highly increased acute phase reactant C-reactive protein (CRP) in the blood. The pathological examination of the synovial membrane identified abundant fibrinous exudate with numerous particles of hyaluronan surrounded by a dense infiltrate of neutrophils and eosinophils. The synovium had moderate hypertrophy and sclerosis as well as an inflammatory infiltrate predominantly composed of T lymphocytes and macrophages with scattered perivascular eosinophils and neutrophils. Immunoperoxidase staining identified numerous deposits of C5b-9 in the fibrinous exudates and the synovial membrane of the patient. Similar findings were observed in the RA patients, whereas deposits were rare in OA synovial samples. In addition, both anaphylatoxin C5a and the terminal complement complex C5b-9 were present at high levels, comparable to those in RA patients. The levels of mRNA for interleukin-1 beta (IL-1ß), IL-6, and the neutrophil marker myeloperoxidase (MPO) were markedly increased compared to those in the RA and OA patients. CONCLUSIONS: This present study is indicative of a pseudo-septic acute inflammatory reaction in response to local accumulation of hylan G-F 20 with the activation of complement and local invasion of pro-inflammatory cells.

Endoglin differentially regulates TGF-ß-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-ß isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-ß receptors. Endoglin is a TGF-ß co-receptor that binds TGF-ß with high affinity in the presence of the type II TGF-ß receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-ß signalling receptors. However, the functional significance of endoglin expression in chondrocytes is unknown. Our objective was to determine whether endoglin regulates TGF-ß/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state. METHOD: Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-ß-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples. RESULTS: Endoglin enhances TGF-ß1-induced Smad1/5 phosphorylation and inhibits TGF-ß1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-ß1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage. CONCLUSION: Together, our results suggest that endoglin regulates the balance between TGF-ß/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype.

Phenotype-related differential alpha-2,6- or alpha-2,3-sialylation of glycoprotein N-glycans in human chondrocytes.

OBJECTIVE: Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either alpha-2,3 or alpha-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans. METHODS: The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) on primary cells were assessed. RESULTS: Primary human chondrocytes predominantly express alpha-2,6-specific SiaTs and accordingly, alpha-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of alpha-2,3-linked sialyl residues and, correspondingly, reduced levels of alpha-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards alpha-2,3-linked sialic acids and alpha-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1beta or tumour necrosis factor-alpha (TNF-alpha). CONCLUSION: The expression of the differentiated chondrocyte phenotype is linked to the ratio of alpha-2,6- to alpha-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.

Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide.

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.

Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism.

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45beta and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-kappaB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

OBJECTIVE: To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS: Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS: At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS: Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Dual proline labeling protocol for individual "baseline" and "response" biosynthesis measurements in human articular cartilage.

OBJECTIVE: The heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol. METHODS: Full-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring (3)H-proline and (14)C-proline incorporation. Each sample's ratio of (14)C-/(3)H-proline incorporation was computed. For comparison to traditional experimental designs, the (14)C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods. RESULTS: The dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): +/-20% (human) and +/-30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their (14)C-proline incorporation (95% CI: +/-50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group. CONCLUSION: The consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.

Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin.

OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes.

In inflammatory diseases such as rheumatoid arthritis, functions of chondrocytes including synthesis of matrix proteins and proteinases are altered through interactions with cells of the infiltrating pannus. One of the major secreted products of mononuclear inflammatory cells is IL-1. In this study we found that recombinant human IL-1 beta suppressed synthesis of cartilage-specific type II collagen by cultured human costal chondrocytes associated with decreased steady state levels of alpha 1 (II) and alpha 1(IX) procollagen mRNAs. In contrast, IL-1 increased synthesis of types I and III collagens and levels of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen mRNAs, as we described previously using human articular chondrocytes and synovial fibroblasts. This stimulatory effect of IL-1 was observed only when IL-1-stimulated PGE2 synthesis was blocked by the cyclooxygenase inhibitor indomethacin. The suppression of type II collagen mRNA levels by IL-1 alone was not due to IL-1-stimulated PGE2, since addition of indomethacin did not reverse, but actually potentiated, this inhibition. Continuous exposure of freshly isolated chondrocytes from day 2 of culture to approximately half-maximal concentrations of IL-1 (2.5 pM) completely suppressed levels of type II collagen mRNA and increased levels of types I and III collagen mRNAs, thereby reversing the ratio of alpha 1(II)/alpha 1(I) procollagen mRNAs from greater than 6.0 to less than 1.0 by day 7. IL-1, therefore, can modify, at a pretranslational level, the relative amounts of the different types of collagen synthesized in cartilage and thereby could be responsible for the inappropriate repair of cartilage matrix in inflammatory conditions.

Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes.

Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4- and chondroitin-6-sulfate. Interleukin-1 beta (IL-1 beta) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1 beta suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1 beta can be used as a model for studying normal and pathological repair mechanisms.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research.

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin-cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

The EWS/TEC fusion protein encoded by the t(9;22) chromosomal translocation in human chondrosarcomas is a highly potent transcriptional activator.

The EWS/TEC gene fusion generated by the t(9;22) chromosomal translocation found in extraskeletal myxoid chondrosarcomas encodes a fusion protein containing the amino-terminal domain of the EWS protein fused to the whole coding sequence of the orphan nuclear receptor TEC. We have compared the DNA-binding and transcriptional activation properties of various TEC isoforms and the corresponding EWS/TEC fusion proteins. Band-shift experiments show that the full-length TEC receptor can efficiently bind the NGFI-B Response Element (NBRE), whereas an isoform lacking the entire carboxyl-terminal domain of the receptor binds much less efficiently the NBRE. Addition of the amino-terminal domain of EWS to either isoforms does not alter significantly their DNA-binding properties to the NBRE. Co-transfection experiments of COS cells and human chondrocytes indicate that whereas TEC moderately activates transcription from a NBRE-containing promoter, the corresponding EWS/TEC fusion protein is a highly potent transcriptional activator of the same promoter, being approximately 270-fold more active than the native receptor. EWS/TEC may thus exert its oncogenic potential in chrondrosarcomas by activating the transcription of target genes involved in cell proliferation.

Human articular chondrocytes acquire 1,25-(OH)2 vitamin D-3 receptors in culture.

The vitamin D endocrine system is crucial in calcium homeostasis in mammalian species. Central to this role 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3) receptors have been detected in freshly isolated osteoblast-like bone cells and it has been shown that the active metabolite of vitamin D-3 1,25-(OH)2D3, increases bone resorption in vitro and in vivo. The requirement of 1,25-(OH)2D3 for the normal development of growth plate cartilage can be seen in vitamin D deficient rickets. However, there is still considerable controversy regarding the presence of 1,25-(OH)2D3 receptors in chondrocytes. In this paper, we report the presence of a 3.5-S 1,25-(OH)2D3-binding macromolecule in freshly isolated human costal but not articular chondrocytes. After subculture, both articular and costal chondrocytes have receptors. Saturation binding analysis revealed a single class of binding sites with an apparent Kd of 0.09 nM and approx. 2700 receptor molecules per cell for articular chondrocytes and a Kd of 0.1 nM and approx. 2000 receptor molecules per cell for costal chondrocytes. The presence of 1,25-(OH)2D3 receptors did not correlate with the switch from synthesis of cartilage-specific type II collagen to types I and III collagens. The acquisition of 1,25-(OH)2D3 receptors by articular chondrocytes may, therefore, be another phenotypic characteristic of cultured cells or may appear in vivo when chondrocytes are exposed to vascular or inflammatory cell products.